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Rajkumar A.P.,University of Aarhus | Rajkumar A.P.,Dementia Clinical Academic Group | Rajkumar A.P.,Kings College | Rajkumar A.P.,The Initiative for Integrative Psychiatric Research | And 17 more authors.
BMC Genomics

Background: Massively parallel cDNA sequencing (RNA-seq) experiments are gradually superseding microarrays in quantitative gene expression profiling. However, many biologists are uncertain about the choice of differentially expressed gene (DEG) analysis methods and the validity of cost-saving sample pooling strategies for their RNA-seq experiments. Hence, we performed experimental validation of DEGs identified by Cuffdiff2, edgeR, DESeq2 and Two-stage Poisson Model (TSPM) in a RNA-seq experiment involving mice amygdalae micro-punches, using high-throughput qPCR on independent biological replicate samples. Moreover, we sequenced RNA-pools and compared their results with sequencing corresponding individual RNA samples. Results: False-positivity rate of Cuffdiff2 and false-negativity rates of DESeq2 and TSPM were high. Among the four investigated DEG analysis methods, sensitivity and specificity of edgeR was relatively high. We documented the pooling bias and that the DEGs identified in pooled samples suffered low positive predictive values. Conclusions: Our results highlighted the need for combined use of more sensitive DEG analysis methods and high-throughput validation of identified DEGs in future RNA-seq experiments. They indicated limited utility of sample pooling strategies for RNA-seq in similar setups and supported increasing the number of biological replicate samples. © 2015 Rajkumar et al. Source

Starnawska A.,University of Aarhus | Starnawska A.,The Initiative for Integrative Psychiatric Research | Demontis D.,University of Aarhus | Demontis D.,The Initiative for Integrative Psychiatric Research | And 13 more authors.
Clinical Epigenetics

Bipolar disorder (BD) and schizophrenia (SZ) are known to share common genetic and psychosocial risk factors. A recent epigenome-wide association study performed on blood samples from SZ patients found significant hypomethylation of FAM63B in exon 9. Here, we used iPLEX-based methylation analysis to investigate two CpG sites in FAM63B in blood samples from 459 BD cases and 268 controls. Both sites were significantly hypomethylated in BD cases (lowest p value = 3.94 × 10−8). The methylation levels at the two sites were correlated, and no strong correlation was found with nearby single nucleotide polymorphisms (SNPs), suggesting that methylation differences at these sites are not readably picked up by genome-wide association studies. Overall, FAM63B hypomethylation was found in BD patients, thus replicating the initial finding in SZ patients. This study suggests that FAM63B is a shared epigenetic risk gene for the two disorders. © 2016, Starnawska et al. Source

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